Gluconic acid is a mild acid with many applications which comprise: its technical use as a complexing agent in industrial cleaning of metal surfaces; its use in the textile industry, in detergents and in concrete; as a food additive in beverages and also in bread and feed; and in pharmaceutical preparations as a chelating agent for ions like Fe. The gluconic acid used in food applications and especially in pharmaceutical preparations must be very pure.
Up until now the production of gluconic acid at industrial scale has been carried out using a fermentation process. Selected microorganisms such as e.g. Aspergillus or Gluconobacter species are grown in a fermenter which is at least equipped with air supply, pH control and temperature control. Optimal conditions are chosen for good growth of the microorganism and optimal development of the enzyme complex capable of converting glucose into gluconic acid/gluconate. At the end of the growth phase, a glucose solution is added to the fermentation broth and aeration is continued. The glucose is converted by the enzymatic complex into gluconic acid. Usually the pH is controlled by the addition of alkali, in which case the majority of gluconic acid will be present as a gluconate salt.
In fungi, glucose is transformed into gluconic acid by an enzymatic complex consisting of glucose oxidase (Gox) and catalase. Glucose oxidase catalyses the reaction: EQU Glucose+O.sub.2 .fwdarw.Gluconolactone+H.sub.2 O.sub.2
The hydrogen peroxide is subsequently split by catalase: EQU H.sub.2 O.sub.2 .fwdarw.H.sub.2 O+1/2O.sub.2
The conversion of gluconolactone into giuconic acid can be catalysed by the enzyme gluconolactonase but can also occur spontaneously: EQU giuconolactone+H.sub.2 O.fwdarw.gluconic acid
Thus, the following overall reaction results:
glucose+1/2O.sub.2 .fwdarw.gluconic acid
The enzymatic complex glucose oxidase/catalase is present in several micro-organisms belonging e.g. to the classes Aspergillus and Penicillium. Also bacteria like Acetobacter and Gluconobacter are capable of converting glucose into gluconic acid but the bacteria use a different mechanism: Gluconobacter oxydans contains two types of glucose dehydrogenases which convert glucose into gluconolactone without the formation of hydrogen peroxide. At this moment, industrial processes for the production of gluconic acid use almost exclusively selected strains of Aspergillus niger or Gluconobacter oxydans in a fermentation process.
The enzymes glucose oxidase and catalase are rather well characterized. The glucose oxidase of A.niger is a glycoprotein with a molecular weight of about 150 kDa and contains prosthetic groups of FAD; the enzyme is active between pH 4 and 7 with an optimal activity at pH 5,5; the enzyme is optimally active at temperatures between 20.degree. and 40.degree. C.; the isoelectric point is about 4,2; the K.sub.m for glucose is 0,11M at 27.degree. C. and the K.sub.m for oxygen is 0.48 mM. The catalase of A.niger has a molecular weight of 250 kDa; the enzyme is active between pH 2 and 7; the optimal temperature is 25.degree. C.; the isoelectric point is about 5,4; the K.sub.m for hydrogen peroxide is about 1,1M or about 37 g/l. The latter means that the affinity of the enzyme catalase for its substrate is very poor.
Taking into account the applications of gluconic acid, the existing fermentative production processes have the following drawbacks. Complex production media are used which, besides glucose, must contain all kinds of nutrients for the growth of the microorganisms, which are costly and give impurities. At the end of the bioconversion the broth contains many byproducts formed during the fermentation such as coloured substances and organic acids other than gluconic acid, such as e.g. citric acid, oxalic acid and 5-keto-gluconic acid. The overall production time includes both the growth time and the bioconversion time and usually requires several days. With fungi, the yield based on glucose is significantly lower than the theoretical maximum yield of 100% because part of the glucose added is used by the microorganism for growth, i.e. for biomass production in stead of gluconic acid production; with Gluconobacter, the yield can be close to 100% but this organisms needs expensive nutrients for growth (W. Olijve, thesis University of Groningen, 1978). Both with fungi and bacteria, at the end of the bioconversion the biomass contained in the broth must be removed. Moreover, especially for the food and pharmaceutical preparations the gluconic acid must undergo extensive further purification in order to remove the above mentioned nutrients and byproducts, which often requires multiple purification steps.
In view of these drawbacks of the fermentative gluconic acid production process, numerous attempts have been made to develop an enzymatic process using the enzymes glucose oxidase and catalase. However, at present no economically feasible enzymatic process for the production of gluconic acid is available. The major reason for this is the low stability of the enzymes involved, even when immobilized. The instability of the enzymes is in fact mainly caused by one of the reaction products, i.e. hydrogen peroxide, which at higher concentrations rapidly inactivates both glucose oxidase and catalase.
The following disclosures exemplify the state of the art of the conversion of glucose into gluconic acid for both fermentation and enzymatic processes:
FR-A-1 590 031 describes a fermentation process for the conversion of glucose in to gluconic acid comprising the stepwise addition of glucose to the fermentation broth. Even though FR-A-1 590 031 suggests that isolated enzymes could also be applied in this process, however, it is not disclosed that this can be done using high glucose concentrations while achieving a conversion rate of more than 50%.
Rosenberg et al. (1992, Bioprocess Eng. 7: 309-313) describe a fermentation process for gluconic acid production in which an a high catalase containing A.niger mutant is used and wherein hydrogen peroxide is applied as oxygen donor. However, as already mentioned, hydrogen peroxide inactivates glucose oxidase and cataiase in isolated form.
Liu et al. (1980, Proc. Natl. Sci. Counc. Repub. China 4: 338-344) use an immobilized enzyme system comprising glucose oxidase and catalase from the fungus Pullularia pullulans to convert glucose solutions of no more than 10% (w/v).
FR-A-2 177 931 discloses an enzyme-system in which catalase and giucose oxidase are immobilized in close proximity of each other in order to protect glucose oxidase from rapid inactivation by hydrogen peroxide. FR-A-2 177 931 teaches to immobilize the enzymes in a catalase/glucose oxidase-activity ratio of at least 1, preferably up to 200. This enzyme system is, however, only used to convert glucose solutions of no more than 5.5% glucose (w/v).
Hartmeier and Doppner (1983, Biotechnol. Lett. 5: 743-748; see also: Doppner and Hartmeier, 1984, Starch 36: 283-287) use the permeabilized mycelium of a glucose oxidase and catalase producing A.niger strain which is coimmobilized with additional catalase to convert glucose into gluconic acid. In this coimmobilized enzyme system catalase and glucose oxidase are present in activity ratios of up to 7600 Baker Units of catalase over 2000 Sarett Units of glucose oxidase (in International cataiase Units this corresponds to about a ratio of about 3950). However, all conversions are carried out with glucose solutions of no more concentrated than 10% (w/v).
RO-92739 describes a process for preparing gluconic acid from glucose in a cyclic process using soluble catalase and glucose oxidase. The glucose concentration used in all examples is 10% (w/v). Catalase/glucose oxidase ratios of up to 200 are used to reach conversion rates of up to 98%.
In a similar cyclic process, Constantinescu et al. (1990, Rev. de Chimie, 41, 5-6: 496-502) test glucose concentrations ranging from 3-20 (w/v). A glucose concentration of 10% (w/v) provides optimal conditions resulting in a conversion rate of 93%. However, when using 15% (w/v) glucose the conversion rate is only about 40%.
FR-A-2 029 645 is concerned with an enzymatic process for the production of gluconic acid in which electrodialysis is used to separate glucose oxidase and the gluconic acid produced and in which hydrogen peroxide is used as oxygen donor. Subsequently fresh glucose is added to restart the reaction. This process is repeated several times. However, a dextrose equivalent of only 100 is used and the yield of gluconic acid is less than 50%.
EP-A-0 017 708 discloses an enzymatic process in which catalase and glucose oxidase are immobilized in a ratio of at least 1:6 in Baker Units of catalase activity and Sarett Units of glucose oxidase activity. The immobilized enzymes are used to convert glucose solutions of 10% and 20% extremely low temperatures: 2.degree. C. and 8.degree. C., respectively. Such low temperatures are, however, not economically feasible at large industrial scale because it would require immense cooling capacity in view of the exothermic nature of the reaction.
Hence, thus far one has either used low glucose concentrations or used low temperatures. Serious drawbacks of the use of low glucose concentrations are, however, that the output (productivity per volume) of the equipment used is low and that diluted gluconic acid solutions are obtained which require energy-intensive removal of the excess water.
There is therefore a need for an enzymatic process for the conversion of glucose into gluconic acid in which high glucose concentrations, i.e. higher than 10% (w/v), can be used, while obtaining a conversion of more than 50% of the glucose used and which can be performed at a temperature of more than 10.degree. C.